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Suppression of feeding, drinking, and locomotion by a putative cannabinoid receptor ‘silent antagonist’
European Journal of Pharmacology 530 (2006) 103 – 106 www.elsevier.com/locate/ejphar
Short communication
Suppression of feeding, drinking, and locomotion by a putative cannabinoid receptor ‘silent antagonist’ Andrew Gardner, Paul E. Mallet ⁎,1 School of Psychology, University of New England, Armidale NSW, 2351, Australia Received 15 September 2005; received in revised form 8 November 2005; accepted 14 November 2005
Abstract This study compared the effects of the putative cannabinoid receptor ‘silent antagonist’ O-2050 with the cannabinoid receptor inverse agonist SR 141716 on food and water consumption, and locomotor activity. Non-deprived male Wistar rats were habituated to the apparatus and testing procedures, then injected intraperitoneally with vehicle, O-2050 (0.03–3.0 mg/kg), or SR 141716 (3.0 mg/kg) prior to 4-h test sessions. Food consumption was significantly reduced by both drugs. Water intake and locomotor activity were significantly reduced only by O-2050. Results support the notion that cannabinoid receptor antagonists suppress feeding behaviour by blocking an endogenous cannabinoid orexigenic signal, rather than by inverse agonism at cannabinoid receptors. However, further studies are needed to confirm the status of O-2050 as a cannabinoid CB1 receptor antagonist devoid of inverse agonist properties. © 2005 Elsevier B.V. All rights reserved. Keywords: Cannabinoid; Feeding behaviour; Motor activity; Drinking behaviour; Body weight; (Rat)
1. Introduction A large body of research supports the notion that appetite is actively regulated by the brain's endocannabinoid system (reviewed by Di Marzo and Matias, 2005). For example, brain concentrations of endogenous cannabinoids rise during fasting and drop during feeding (Kirkham et al., 2002), and cannabinoid CB1 receptor knockout (CB1-KO) mice show reduced food consumption (Di Marzo et al., 2001) compared to wild type mice following brief food restriction. Food consumption is also potentiated by administration of Δ9-tetrahydrocannabinol (e.g., Verty et al., 2004c) and endogenous cannabinoids such as anandamide (Williams and Kirkham, 1999). Moreover, the administration of the
⁎ Corresponding author. Tel.: +61 2 6773 3725; fax: +61 2 6773 3820. E-mail address: [email protected] (P.E. Mallet). 1 Note: From January 1, 2006, Dr. Mallet's mailing address will be Department of Psychology, Wilfrid Laurier University, Sciences Building, 75 University Ave., West Waterloo, Ontario, N2L 3C5, Canada. The E-mail address will remain the same. 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.11.032
cannabinoid CB1 receptor antagonist SR 141716 (rimonabant) reverses cannabinoid agonist-induced hyperphagia (Williams and Kirkham, 1999) and attenuates basal feeding (Colombo et al., 1998), suggesting that cannabinoid CB1 receptor antagonists may have some utility in the treatment of obesity (Van Gaal et al., 2005). The majority of cannabinoid CB1 receptor antagonists developed thus far are not pure antagonists; most also possess inverse agonist properties across a range of in vitro and in vivo preparations (reviewed by Pertwee, 2005). It therefore remains unclear if the anorectic effects of cannabinoid receptor antagonists can be attributed to the mere blockade of cannabinoid CB1 receptors, or to the negative modulation of the constitutive receptor activity (i.e., inverse agonism). Recently, a sulphonamide analogue of Δ8-tetrahydrocannabinol possessing high affinity for CB1 cannabinoid receptors was developed. Called O-2050, this putative “silent antagonist” possesses no agonist or inverse agonist activity in both the guinea pig myenteric longitudinal muscle, and mouse isolated vas deferens, preparations (reviewed by Pertwee, 2005). The present study therefore sought to determine whether the
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feeding-suppressive effects of cannabinoid receptor antagonists are related to their inverse agonist properties by comparing the effects of O-2050 and SR 141716 on food consumption. To further characterise the in vivo effects of O-2050, other measures included water intake, locomotor activity, and body weight. 2. Materials and methods Animals were treated in accordance with the “Principles of laboratory animal care” (NIH publication no. 85-23, revised 1985) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. This study was reviewed and approved by the University of New England Animal Ethics Committee. 2.1. Subjects Experimentally naïve male albino Wistar rats, aged 7–8 weeks, were used. Rats were housed 5–6 per group as previously described (Verty et al., 2003) in a climate-controlled room on a 12:12 h reverse light:dark cycle (lights off at 0800 h). Testing commenced at 0830 h. Rats had ad libitum access to laboratory chow (Rat and mouse chow, Ridley AgriProducts, Australia) and tap water while in their home cages. 2.2. Drugs O-2050 ((6aR,10aR)-3-(1-Methanesulfonylamino-4-hexyn6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d] pyran; Tocris, UK) and SR 141716 (N-piperidino-5-(4-chlorophenyl)-1-)2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide; Sanofi Recherche, Montpellier, France) were dissolved in a vehicle consisting of 0.9% (v/v) TWEEN 80 in saline (0.9% w/v) as described previously for SR 141716 (Verty et al., 2003). SR 141716 (3.0 mg/kg) and O-2050 (0.03, 0.3, and 3.0 mg/kg) were administered intraperitoneally in a volume of 1 ml/kg.
and water intake were measured by weight each hour. Next, rats received additional “test sessions” in which they were injected with vehicle (0.9% v/v TWEEN 80 in 0.9% w/v saline), O2050, or SR 141716, prior to each 4 h test. The amount of food and water consumed were again measured hourly. Locomotor activity (time in motion) was recorded and grouped into four 1h “bins” for statistical analysis. Drug tests were conducted every 48 h and all rats received all treatments (five in total) in a counter-balanced order. On days between drug tests animals were tested as usual, but no drugs were administered. To determine the short-term effects of drug administration on body weight, rats were weighed 24 h after administration of drug or vehicle. 2.5. Statistical analysis The amount of food consumed (g), water consumed (ml), time spent in motion (s) and body weight (g), were used as dependant variables and were analysed separately using twofactor repeated measures (treatment by time) analysis of variance (ANOVA). Body weight data were analysed using a one-way repeated measures ANOVA. Where significant main effects were found, pairwise comparisons were conducted using Bonferroni-adjusted t-tests. The five (α = 0.01) comparisons of interest were: (1) Vehicle vs. SR 141716, (2) Vehicle vs. 0.03 mg/kg O-2050, (3) Vehicle vs. 0.3 mg/kg O-2050, (4) Vehicle vs. 3.0 mg/kg O-2050, and (5) SR 141716 vs. 3.0 mg/kg O-2050. Significant interactions were followed by one-way tests of the simple main effects, and Bonferroni-adjusted t-test (as above) when these were significant. 3. Results 3.1. Food consumption
The experiment was conducted in eight identical dimly lit (13 lx) sound-attenuated chambers as previously described (Verty et al., 2004d). The test food (rat and mouse chow, Ridley AgriProducts, Australia) was presented in glass dishes. Plastic drinking bottles containing tap water were available in the test chambers. A computer monitored passive infrared detectors mounted to the ceiling of each box measure the time spent in motion (Verty et al., 2003).
Baseline feeding was fairly consistent across the four 1-h measurement intervals following vehicle treatment (Fig. 1, top). Both SR 141716 and 3.0 mg/kg O-2050 significantly reduced food consumption. Two-way ANOVA revealed a significant main effect of treatment, [F(4, 56) = 21.64, P b 0.001], main effect of time, [F(3, 42) = 8.34, P b 0.001], and treatment by time interaction, [F(12, 168) = 4.13, P b 0.001]. One-way ANOVAs conducted at each time interval revealed a significant effect of treatment at 1 h [F(4, 56) = 15.52, P b 0.001], 3 h [F(4, 56) = 1.82, P b 0.05] and 4 h [F(4, 56) = 4.69, P b 0.01], but not at 2 h. Pairwise comparisons revealed that feeding was significantly reduced by 3.0 mg/kg O-2050 and SR 141716 at hour 1 only. Feeding was significantly reduced by 0.3 mg/kg O-2050 at 3 h only.
2.4. Procedure
3.2. Water intake
Rats (n = 16) were tested every 24 h at approximately the same time each day. For the first 4 days, animals were placed in the activity chambers with 100 g laboratory chow and 300 ml water. During these “habituation sessions”, rats were injected with saline (1 ml/kg) and placed in the apparatus for 4 h; food
Water intake was significantly reduced by 3.0 mg/kg O-2050 (Fig. 1, middle). SR 141716 and 0.3 mg/kg O-2050 reduced water intake slightly, but not significantly. The main effects of treatment [F(4, 56) = 7.93, P b 0.001], and time [F(3, 42) = 11.73, P b 0.001], were significant, but the interaction was not.
2.3. Apparatus
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3.3. Locomotor activity
Table 1 Change in body weight 24 h following drug administration
The two highest doses of O-2050 significantly reduced locomotor activity relative to vehicle (Fig. 1, bottom). Furthermore, administration of 3.0 mg/kg O-2050 significantly reduced locomotor activity compared to SR 141716. Two-way ANOVA revealed a significant main effect of treatment, [F(4, 56) = 27.33, P b 0.001], main effect of time, [F(3, 42) = 30.35, P b 0.001], and treatment by time interaction, [F(12, 168) = 3.66, P b 0.001]. Tests of the simple main effects revealed significant effects of treatment at 1 h [F(4, 56) = 7.30, P b 0.001], 2 h [F(4, 56) = 8.57, P b 0.001], 3 h [F(4, 56) = 21.79, P b 0.001] and 4 h [F(4, 56) = 22.24, P b 0.001]. At all measurement intervals, 3.0 mg/kg O-2050 significantly reduced locomotor activity compared to vehicle and SR 141716. Furthermore, 0.3 mg/kg O-2050 significantly reduced locomotion at 3 and 4 h only.
Treatment
Weight gain (g ± S.E.M.)
Vehicle 0.03 mg/kg O-2050 0.3 mg/kg O-2050 3.0 mg/kg O-2050 3.0 mg/kg SR 141716
4.88 ± 1.11 3.69 ± 0.87 2.69 ± 1.06 1.13 ± 0.81 −0.25 ± 1.74
3.4. Body weight Administration of O-2050 and SR 141716 reduced body weight relative to vehicle (Table 1). The one-way ANOVA was significant [F(4, 60) = 3.13, P b 0.05]; however, the Bonferroniadjusted (α = 0.01) pairwise comparisons revealed no significant differences between doses, although SR 141716 and 3.0 mg/kg O-2050 approached significance (P = 0.018 and 0.021, respectively). 4. Discussion
Fig. 1. Laboratory chow consumed (top), water intake (middle), and locomotor activity (bottom) in non-deprived rats after IP administration of vehicle, 3.0 mg/kg SR 141716, or O-2050 (0.03, 0.3, or 3.0 mg/kg). Data represent means (+ S.E.M.) at four consecutive 1-h measurement intervals. *Significantly different from Vehicle; †significantly different from SR 141716.
Results revealed that similar doses of the putative cannabinoid receptor silent antagonist O-2050 and the cannabinoid inverse agonist SR 141716 equally suppressed food consumption. These findings confirm the involvement of the brain's endocannabinoid system in feeding behaviour, and support the notion that appetite is actively regulated by an endocannabinoid signal. These findings also suggest that the anorectic effects of SR 141716 (e.g., Verty et al., 2004d) and other cannabinoid receptor antagonists such as AM 281 (Werner and Koch, 2003) are not related to the inverse agonist properties of these compounds, but are due to functional opposition of endocannabinoids. This is in accord with the finding that CB1-KO mice do not respond to the anorectic effect of SR 141716 and consume less food relative to wild type control animals (Di Marzo et al., 2001). Studies have shown that cannabinoid receptor agonists often inhibit spontaneous locomotion and induce catalepsy, or produce biphasic effects on locomotor activity (reviewed by Sañudo-Peña et al., 2000). Cannabinoid receptor antagonists such as SR 141716 typically do not influence locomotor activity (e.g., Verty et al., 2004a,b,d), but results from studies using CB1-KO mice have been mixed with one study showing normal activity, and another reporting hyperactivity in CB1-KO mice (reviewed by Sañudo-Peña et al., 2000). It is therefore somewhat surprising that O-2050 induced significant hypoactivity in the present study. The reasons for this remain unclear, but it may be related to some unidentified non-cannabinoid action of O-2050. Regardless of the mechanism(s) at play, the reduction of activity by O-2050 complicates the interpretation of the food consumption data. That is, it is not possible to determine if O2050 reduced feeding by suppressing appetite or by reducing motivation in general. O-2050 also reduced water consumption, although it is not clear if this was due to a reduction of thirst, a general sedative effect of the drug, or a reduced need for water due to diminished chow intake. It is, however, worth noting that
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the time courses for the feeding and locomotor effects of O-2050 were dissimilar (cf. Fig. 1, top and bottom), suggesting that locomotor suppression was not solely responsible for the reduction in feeding. There is growing interest in the therapeutic potential of cannabinoid receptor antagonists in weight control. Indeed SR 141716 (rimonabant), marketed as ‘Acomplia’ by SanofiAventis, has progressed to third phase clinical trials. Results from a recent multinational randomised placebo-controlled trial revealed that daily treatment with 20 mg rimonabant for 1 year led to significant reductions in body weight and waist circumference, and various improvements in cardiovascular risk factors (Van Gaal et al., 2005). However, given the similar dose–responses of O-2050 for food consumption and locomotor activity found here, a clinically effective dose of this compound may not be free from significant side effects. We have not characterized the effects of O-2050 on feeding in food-restricted animals, but this remains an important direction for future research. Limbic forebrain and hypothalamic endocannabinoid levels rise in food-restricted, but not in satiated rats (Kirkham et al., 2002). Because satiated rats were used in the present study, the endocannabinoid tone targeted by O-2050 may have been far from maximal, or perhaps even absent. Whether O-2050 can attenuate feeding without influencing locomotor activity in food-restricted animals remains to be seen. In summary, O-2050 and SR 141716 were found to be roughly equipotent in suppressing feeding behaviour, but unlike SR 141716, O-2050 also decreased spontaneous locomotion. We conclude from the present findings and recent studies using CB1-KO mice that cannabinoid receptor antagonists suppress feeding by blocking an endogenous cannabinoid orexigenic signal, rather than by inverse agonism at cannabinoid receptors. However, because little pharmacological data are available for O-2050, these conclusions are tentative until further studies confirm the status of this compound as a specific cannabinoid CB1 receptor antagonist devoid of inverse agonist properties. Acknowledgements Funding provided by a grant from the University of New England to P.E.M. We are grateful to Sanofi Recherche for
supplying SR 141716, and Dr. Aaron Verty for comments on an early manuscript draft. References Colombo, G.R., Agabio, R., Diaz, G., Lobina, C., Reali, R., Gessa, G.L., 1998. Appetite suppression and weight loss after the cannabinoid antagonist SR141716. Life Sci. 63, PL113–PL117. Di Marzo, V., Matias, I., 2005. Endocannabinoid control of food intake and energy balance. Nat. Neurosci. 8, 585–589. Di Marzo, V., Goparaju, S.K., Wang, L., Liu, J., Batkai, S., Jarai, Z., Fezza, F., Miura, G.I., Palmiter, R.D., Sugiura, T., Kunos, G., 2001. Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature 410, 822–825. Kirkham, T.C., Williams, C.M., Fezza, F., Di Marzo, V., 2002. Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br. J. Pharmacol. 136, 550–557. Pertwee, R.G., 2005. Inverse agonism and neutral antagonism at cannabinoid CB1 receptors. Life Sci. 76, 1307–1324. Sañudo-Peña, M.C., Romero, J., Seale, G.E., Fernandez-Ruiz, J., Walker, J.M., 2000. Activational role of cannabinoids on movement. Eur. J. Pharmacol. 391, 269–274. Van Gaal, L.F., Rissanen, A.M., Scheen, A.J., Ziegler, O., Rossner, S., 2005. Effects of the cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight patients: 1-year experience from the RIO-Europe study. Lancet 365, 1389–1397. Verty, A.N.A., Singh, M.E., McGregor, I.S., Mallet, P.E., 2003. The cannabinoid receptor antagonist SR 141716 attenuates overfeeding induced by systemic or intracranial morphine. Psychopharmacology 168, 314–323. Verty, A.N.A., McFarlane, J.R., McGregor, I.S., Mallet, P.E., 2004a. Evidence for an interaction between CB1 cannabinoid and melanocortin MCR-4 receptors in regulating food intake. Endocrinology 145, 3224–3231. Verty, A.N.A., McFarlane, J.R., McGregor, I.S., Mallet, P.E., 2004b. Evidence for an interaction between CB1 cannabinoid and oxytocin receptors in food and water intake. Neuropharmacology 47, 593–603. Verty, A.N.A., McGregor, I.S., Mallet, P.E., 2004c. The dopamine receptor antagonist SCH 23390 attenuates feeding induced by Δ9-tetrahydrocannabinol. Brain Res. 1020, 188–195. Verty, A.N.A., McGregor, I.S., Mallet, P.E., 2004d. A cannabinoid receptor antagonist in non-deprived rats equally suppresses consumption of high carbohydrate, high fat, and normal chow. Neurosci. Lett. 354, 217–220. Werner, N.A., Koch, J.E., 2003. Effects of the cannabinoid antagonists AM281 and AM630 on deprivation-induced intake in Lewis rats. Brain Res. 967, 290–292. Williams, C.M., Kirkham, T.C., 1999. Anandamide induces overeating: mediation by central cannabinoid (CB1) receptors. Psychopharmacology 143, 315–317.
Short communication
Suppression of feeding, drinking, and locomotion by a putative cannabinoid receptor ‘silent antagonist’ Andrew Gardner, Paul E. Mallet ⁎,1 School of Psychology, University of New England, Armidale NSW, 2351, Australia Received 15 September 2005; received in revised form 8 November 2005; accepted 14 November 2005
Abstract This study compared the effects of the putative cannabinoid receptor ‘silent antagonist’ O-2050 with the cannabinoid receptor inverse agonist SR 141716 on food and water consumption, and locomotor activity. Non-deprived male Wistar rats were habituated to the apparatus and testing procedures, then injected intraperitoneally with vehicle, O-2050 (0.03–3.0 mg/kg), or SR 141716 (3.0 mg/kg) prior to 4-h test sessions. Food consumption was significantly reduced by both drugs. Water intake and locomotor activity were significantly reduced only by O-2050. Results support the notion that cannabinoid receptor antagonists suppress feeding behaviour by blocking an endogenous cannabinoid orexigenic signal, rather than by inverse agonism at cannabinoid receptors. However, further studies are needed to confirm the status of O-2050 as a cannabinoid CB1 receptor antagonist devoid of inverse agonist properties. © 2005 Elsevier B.V. All rights reserved. Keywords: Cannabinoid; Feeding behaviour; Motor activity; Drinking behaviour; Body weight; (Rat)
1. Introduction A large body of research supports the notion that appetite is actively regulated by the brain's endocannabinoid system (reviewed by Di Marzo and Matias, 2005). For example, brain concentrations of endogenous cannabinoids rise during fasting and drop during feeding (Kirkham et al., 2002), and cannabinoid CB1 receptor knockout (CB1-KO) mice show reduced food consumption (Di Marzo et al., 2001) compared to wild type mice following brief food restriction. Food consumption is also potentiated by administration of Δ9-tetrahydrocannabinol (e.g., Verty et al., 2004c) and endogenous cannabinoids such as anandamide (Williams and Kirkham, 1999). Moreover, the administration of the
⁎ Corresponding author. Tel.: +61 2 6773 3725; fax: +61 2 6773 3820. E-mail address: [email protected] (P.E. Mallet). 1 Note: From January 1, 2006, Dr. Mallet's mailing address will be Department of Psychology, Wilfrid Laurier University, Sciences Building, 75 University Ave., West Waterloo, Ontario, N2L 3C5, Canada. The E-mail address will remain the same. 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.11.032
cannabinoid CB1 receptor antagonist SR 141716 (rimonabant) reverses cannabinoid agonist-induced hyperphagia (Williams and Kirkham, 1999) and attenuates basal feeding (Colombo et al., 1998), suggesting that cannabinoid CB1 receptor antagonists may have some utility in the treatment of obesity (Van Gaal et al., 2005). The majority of cannabinoid CB1 receptor antagonists developed thus far are not pure antagonists; most also possess inverse agonist properties across a range of in vitro and in vivo preparations (reviewed by Pertwee, 2005). It therefore remains unclear if the anorectic effects of cannabinoid receptor antagonists can be attributed to the mere blockade of cannabinoid CB1 receptors, or to the negative modulation of the constitutive receptor activity (i.e., inverse agonism). Recently, a sulphonamide analogue of Δ8-tetrahydrocannabinol possessing high affinity for CB1 cannabinoid receptors was developed. Called O-2050, this putative “silent antagonist” possesses no agonist or inverse agonist activity in both the guinea pig myenteric longitudinal muscle, and mouse isolated vas deferens, preparations (reviewed by Pertwee, 2005). The present study therefore sought to determine whether the
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feeding-suppressive effects of cannabinoid receptor antagonists are related to their inverse agonist properties by comparing the effects of O-2050 and SR 141716 on food consumption. To further characterise the in vivo effects of O-2050, other measures included water intake, locomotor activity, and body weight. 2. Materials and methods Animals were treated in accordance with the “Principles of laboratory animal care” (NIH publication no. 85-23, revised 1985) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. This study was reviewed and approved by the University of New England Animal Ethics Committee. 2.1. Subjects Experimentally naïve male albino Wistar rats, aged 7–8 weeks, were used. Rats were housed 5–6 per group as previously described (Verty et al., 2003) in a climate-controlled room on a 12:12 h reverse light:dark cycle (lights off at 0800 h). Testing commenced at 0830 h. Rats had ad libitum access to laboratory chow (Rat and mouse chow, Ridley AgriProducts, Australia) and tap water while in their home cages. 2.2. Drugs O-2050 ((6aR,10aR)-3-(1-Methanesulfonylamino-4-hexyn6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d] pyran; Tocris, UK) and SR 141716 (N-piperidino-5-(4-chlorophenyl)-1-)2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide; Sanofi Recherche, Montpellier, France) were dissolved in a vehicle consisting of 0.9% (v/v) TWEEN 80 in saline (0.9% w/v) as described previously for SR 141716 (Verty et al., 2003). SR 141716 (3.0 mg/kg) and O-2050 (0.03, 0.3, and 3.0 mg/kg) were administered intraperitoneally in a volume of 1 ml/kg.
and water intake were measured by weight each hour. Next, rats received additional “test sessions” in which they were injected with vehicle (0.9% v/v TWEEN 80 in 0.9% w/v saline), O2050, or SR 141716, prior to each 4 h test. The amount of food and water consumed were again measured hourly. Locomotor activity (time in motion) was recorded and grouped into four 1h “bins” for statistical analysis. Drug tests were conducted every 48 h and all rats received all treatments (five in total) in a counter-balanced order. On days between drug tests animals were tested as usual, but no drugs were administered. To determine the short-term effects of drug administration on body weight, rats were weighed 24 h after administration of drug or vehicle. 2.5. Statistical analysis The amount of food consumed (g), water consumed (ml), time spent in motion (s) and body weight (g), were used as dependant variables and were analysed separately using twofactor repeated measures (treatment by time) analysis of variance (ANOVA). Body weight data were analysed using a one-way repeated measures ANOVA. Where significant main effects were found, pairwise comparisons were conducted using Bonferroni-adjusted t-tests. The five (α = 0.01) comparisons of interest were: (1) Vehicle vs. SR 141716, (2) Vehicle vs. 0.03 mg/kg O-2050, (3) Vehicle vs. 0.3 mg/kg O-2050, (4) Vehicle vs. 3.0 mg/kg O-2050, and (5) SR 141716 vs. 3.0 mg/kg O-2050. Significant interactions were followed by one-way tests of the simple main effects, and Bonferroni-adjusted t-test (as above) when these were significant. 3. Results 3.1. Food consumption
The experiment was conducted in eight identical dimly lit (13 lx) sound-attenuated chambers as previously described (Verty et al., 2004d). The test food (rat and mouse chow, Ridley AgriProducts, Australia) was presented in glass dishes. Plastic drinking bottles containing tap water were available in the test chambers. A computer monitored passive infrared detectors mounted to the ceiling of each box measure the time spent in motion (Verty et al., 2003).
Baseline feeding was fairly consistent across the four 1-h measurement intervals following vehicle treatment (Fig. 1, top). Both SR 141716 and 3.0 mg/kg O-2050 significantly reduced food consumption. Two-way ANOVA revealed a significant main effect of treatment, [F(4, 56) = 21.64, P b 0.001], main effect of time, [F(3, 42) = 8.34, P b 0.001], and treatment by time interaction, [F(12, 168) = 4.13, P b 0.001]. One-way ANOVAs conducted at each time interval revealed a significant effect of treatment at 1 h [F(4, 56) = 15.52, P b 0.001], 3 h [F(4, 56) = 1.82, P b 0.05] and 4 h [F(4, 56) = 4.69, P b 0.01], but not at 2 h. Pairwise comparisons revealed that feeding was significantly reduced by 3.0 mg/kg O-2050 and SR 141716 at hour 1 only. Feeding was significantly reduced by 0.3 mg/kg O-2050 at 3 h only.
2.4. Procedure
3.2. Water intake
Rats (n = 16) were tested every 24 h at approximately the same time each day. For the first 4 days, animals were placed in the activity chambers with 100 g laboratory chow and 300 ml water. During these “habituation sessions”, rats were injected with saline (1 ml/kg) and placed in the apparatus for 4 h; food
Water intake was significantly reduced by 3.0 mg/kg O-2050 (Fig. 1, middle). SR 141716 and 0.3 mg/kg O-2050 reduced water intake slightly, but not significantly. The main effects of treatment [F(4, 56) = 7.93, P b 0.001], and time [F(3, 42) = 11.73, P b 0.001], were significant, but the interaction was not.
2.3. Apparatus
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3.3. Locomotor activity
Table 1 Change in body weight 24 h following drug administration
The two highest doses of O-2050 significantly reduced locomotor activity relative to vehicle (Fig. 1, bottom). Furthermore, administration of 3.0 mg/kg O-2050 significantly reduced locomotor activity compared to SR 141716. Two-way ANOVA revealed a significant main effect of treatment, [F(4, 56) = 27.33, P b 0.001], main effect of time, [F(3, 42) = 30.35, P b 0.001], and treatment by time interaction, [F(12, 168) = 3.66, P b 0.001]. Tests of the simple main effects revealed significant effects of treatment at 1 h [F(4, 56) = 7.30, P b 0.001], 2 h [F(4, 56) = 8.57, P b 0.001], 3 h [F(4, 56) = 21.79, P b 0.001] and 4 h [F(4, 56) = 22.24, P b 0.001]. At all measurement intervals, 3.0 mg/kg O-2050 significantly reduced locomotor activity compared to vehicle and SR 141716. Furthermore, 0.3 mg/kg O-2050 significantly reduced locomotion at 3 and 4 h only.
Treatment
Weight gain (g ± S.E.M.)
Vehicle 0.03 mg/kg O-2050 0.3 mg/kg O-2050 3.0 mg/kg O-2050 3.0 mg/kg SR 141716
4.88 ± 1.11 3.69 ± 0.87 2.69 ± 1.06 1.13 ± 0.81 −0.25 ± 1.74
3.4. Body weight Administration of O-2050 and SR 141716 reduced body weight relative to vehicle (Table 1). The one-way ANOVA was significant [F(4, 60) = 3.13, P b 0.05]; however, the Bonferroniadjusted (α = 0.01) pairwise comparisons revealed no significant differences between doses, although SR 141716 and 3.0 mg/kg O-2050 approached significance (P = 0.018 and 0.021, respectively). 4. Discussion
Fig. 1. Laboratory chow consumed (top), water intake (middle), and locomotor activity (bottom) in non-deprived rats after IP administration of vehicle, 3.0 mg/kg SR 141716, or O-2050 (0.03, 0.3, or 3.0 mg/kg). Data represent means (+ S.E.M.) at four consecutive 1-h measurement intervals. *Significantly different from Vehicle; †significantly different from SR 141716.
Results revealed that similar doses of the putative cannabinoid receptor silent antagonist O-2050 and the cannabinoid inverse agonist SR 141716 equally suppressed food consumption. These findings confirm the involvement of the brain's endocannabinoid system in feeding behaviour, and support the notion that appetite is actively regulated by an endocannabinoid signal. These findings also suggest that the anorectic effects of SR 141716 (e.g., Verty et al., 2004d) and other cannabinoid receptor antagonists such as AM 281 (Werner and Koch, 2003) are not related to the inverse agonist properties of these compounds, but are due to functional opposition of endocannabinoids. This is in accord with the finding that CB1-KO mice do not respond to the anorectic effect of SR 141716 and consume less food relative to wild type control animals (Di Marzo et al., 2001). Studies have shown that cannabinoid receptor agonists often inhibit spontaneous locomotion and induce catalepsy, or produce biphasic effects on locomotor activity (reviewed by Sañudo-Peña et al., 2000). Cannabinoid receptor antagonists such as SR 141716 typically do not influence locomotor activity (e.g., Verty et al., 2004a,b,d), but results from studies using CB1-KO mice have been mixed with one study showing normal activity, and another reporting hyperactivity in CB1-KO mice (reviewed by Sañudo-Peña et al., 2000). It is therefore somewhat surprising that O-2050 induced significant hypoactivity in the present study. The reasons for this remain unclear, but it may be related to some unidentified non-cannabinoid action of O-2050. Regardless of the mechanism(s) at play, the reduction of activity by O-2050 complicates the interpretation of the food consumption data. That is, it is not possible to determine if O2050 reduced feeding by suppressing appetite or by reducing motivation in general. O-2050 also reduced water consumption, although it is not clear if this was due to a reduction of thirst, a general sedative effect of the drug, or a reduced need for water due to diminished chow intake. It is, however, worth noting that
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the time courses for the feeding and locomotor effects of O-2050 were dissimilar (cf. Fig. 1, top and bottom), suggesting that locomotor suppression was not solely responsible for the reduction in feeding. There is growing interest in the therapeutic potential of cannabinoid receptor antagonists in weight control. Indeed SR 141716 (rimonabant), marketed as ‘Acomplia’ by SanofiAventis, has progressed to third phase clinical trials. Results from a recent multinational randomised placebo-controlled trial revealed that daily treatment with 20 mg rimonabant for 1 year led to significant reductions in body weight and waist circumference, and various improvements in cardiovascular risk factors (Van Gaal et al., 2005). However, given the similar dose–responses of O-2050 for food consumption and locomotor activity found here, a clinically effective dose of this compound may not be free from significant side effects. We have not characterized the effects of O-2050 on feeding in food-restricted animals, but this remains an important direction for future research. Limbic forebrain and hypothalamic endocannabinoid levels rise in food-restricted, but not in satiated rats (Kirkham et al., 2002). Because satiated rats were used in the present study, the endocannabinoid tone targeted by O-2050 may have been far from maximal, or perhaps even absent. Whether O-2050 can attenuate feeding without influencing locomotor activity in food-restricted animals remains to be seen. In summary, O-2050 and SR 141716 were found to be roughly equipotent in suppressing feeding behaviour, but unlike SR 141716, O-2050 also decreased spontaneous locomotion. We conclude from the present findings and recent studies using CB1-KO mice that cannabinoid receptor antagonists suppress feeding by blocking an endogenous cannabinoid orexigenic signal, rather than by inverse agonism at cannabinoid receptors. However, because little pharmacological data are available for O-2050, these conclusions are tentative until further studies confirm the status of this compound as a specific cannabinoid CB1 receptor antagonist devoid of inverse agonist properties. Acknowledgements Funding provided by a grant from the University of New England to P.E.M. We are grateful to Sanofi Recherche for
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